Multi-line coherent Raman imaging Coherent Raman microscopy, by merging coherent Raman vibrational spectroscopy with optical microscopy, has been demonstrated as a powerful platform for label free imaging. However, traditional coherent Raman imaging techniques usually fall into one of the two categories: single line vs. broadband. The former is fast but has limited specificity, while the latter provides a wealth of spectral information and thus has high specificity, but suffers from much slower imaging speed. To address these challenges, we propose a multi-line coherent Raman imaging method that essentially splits the difference between single line and broadband coherent Raman, allowing for simultaneous excitation of multiple vibrational peaks in order to achieve both high specificity and high speed imaging. The key enabling technical innovation is our method of coupling pulse division and recombination techniques with the soliton self frequency shift to achieve a fiber-based, power scalable, broadly tunable, multi-color ultrashort excitation source, which makes the practical implementation of the proposed imaging methodology possible. In this proposed program, we will first develop a Yb:fiber laser based divided pulse soliton self frequency shift source, which can allow the simultaneous excitation of four vibrational lines. Next, we will develop a multi-line coherent Raman imaging system by using the developed multi-color source. We will perform systematic characterization and optimization to meet the design goals, and use the system to monitor synapse formation in the developing nigrostriatal pathway in vitro in order to demonstrate the power of the proposed imaging technique for enabling new and important studies of dynamic processes in brain tissues in their natural state.